A case of onychomycosis caused by Aspergillus candidus

Medical Mycology Case Reports 1 (2012) 45–48 Contents lists available at SciVerse ScienceDirect Medical Mycology Case Reports journal homepage: www.elsevier.com/locate/mmcr A case of onychomycosis caused by Aspergillus candidus Bahram Ahmadi a, Seyed Jamal Hashemi a, Farideh Zaini a, Mohammad Reza Shidfar a, Maryam Moazeni a, Bita Mousavi b, Fatemeh Noorbakhsh c, Mohsen Gheramishoar a, Leila Hossein pour a, Sassan Rezaie d,n a Division of Molecular Biology, Department of Medical Mycology & Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran c Department of Biology, Islamic Azad University, Varamin-Pishva Branch, Varamin, Iran d Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran b a r t i c l e i n f o abstract Article history: Received 9 June 2012 Received in revised form 25 June 2012 Accepted 27 June 2012 Based on epidemiological studies, Aspergillus candidus has been demonstrated as an emerging fungal agent of toenail onychomycosis. Here we report a case of a toenail infection caused by A. candidus in a healthy 60-year-old woman. Based on macroscopic and microscopic characteristics of the culture as well as nucleotide sequencing of 28S region, the causative agent was identified as A. candidus. & 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights reserved. Keywords: Aspergillus candidus Onychomycosis DNA sequencing 1. Introduction Onychomycosis is a frequent fungal disease in nails which is triggered by dermatophytes, yeasts and non-dermatophyte molds (NDMs) [1]. Although non-dermatophyte filamentous fungi are commonly found in soil as well as plant debris and mostly considered as plant pathogens, they are also known as causing agents of onychomycosis and therefore, are the aim of considerable studies in this field [2,3]. Due to various socioeconomic and cultural factors, the prevalence of onychomycosis varies globally. Moreover, these infections are however depending on the population, geographic area and the mycological or diagnostic methods used. They are more common in people over 60 years of age [4,5]. Nevertheless, onychomycosis affects 3–26% of people worldwide, which shows this infection as a significant health problem [6]. The main NDM species regarded as causative agents of onychomycosis are as follows: Scopulariopsis, Scytalidium, Fusarium, Aspergillus and Onychocola canadensis [7]. Unlike dermatophytes, NDMs are usually not keratinolytic. For this reason, a previous keratin destruction by dermatophytes, or trauma as well as n Correspondence to: Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, No. 88, Italy Street, Keshavarz Boulevard, P.O. Box 14155-6446, Tehran, Iran. Tel.: þ98 21 88 95 13 92; fax: þ98 21 66 46 22 67. E-mail address: [email protected] (S. Rezaie). another nail disease, can provide the NDMs a perfect condition to invade the nail plate. Therefore, they are usually found as secondary invaders but not as primary parasites of the nail plate [8]. In the present study, we describe a case of onychomycosis caused by Aspergillus candidus in Iran. 2. Case On February 2010, a 60-year-old house wife with a suspected onychomycosis of her right great toenail was referred to the laboratory of Medical Mycology in Tehran University of Medical Sciences (Day 0). She had a significant discoloration with a subungual hyperkeratosis that caused color alteration and rough surface areas in the mentioned nail. However, the nail plate remained substantially intact from the structural point of view (Fig. 1). The patient was in good health and despite her notification about the nail discoloration, she had ignored it for about 6 years. Other nails as well as the skin of the soles and interdigital webs were normal. No history of trauma or nail abnormalities prior to the lesion was mentioned by this patient. Direct microscopic examination of nail samples with 15% KOH showed rather broad septate and irregular hyphae which did not correspond to the hyphae and regular chains of arthroconidia produced in tissues by dermatophytes (Fig. 2). Portions of the nail were inoculated on Sabouraud Dextrose Agar medium (Merck, Germany), supplied with chloramphenicol (SC), on several different locations of plate. A slowly-grown cream colony was yielded 2211-7539/$ - see front matter & 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2012.06.003 46 B. Ahmadi et al. / Medical Mycology Case Reports 1 (2012) 45–48 Fig. 1. Clinical features of the toenail lesion caused by Aspergillus candidus. A total discolouration with a subungual hyperkeratosis is obviously observed. Fig. 2. 15% KOH slide preparation of the nail scraping showed rather broad septate and irregular hyphae (  400). from all inoculated points which, according to macroscopic and microscopic examinations, was identified as a member of the genus Aspergillus. Consequently, inoculation of portions of the colony on Czapek’s agar (Difco, USA) yielded a slowly-grown pale yellow colony. The macroscopic and microscopic morphology revealed that the causative agent of this infection is likely to be A. candidus based on the characteristics described previously [9]. Typically, the fungus possesses white as well as globose conidiaproducing bodies which produce globose and subglobose conidia. These conidia were smooth, thin-walled and revealed to be about 2.5–3.5 mm in diameter. The mentioned isolate has spherical to subspherical vesicles which were entirely covered with metulae. Besides, molecular identification was done in order to confirm the morphological identification (Fig. 3). Fungal DNA was directly extracted from the affected nail sample using Qiagen DNeasy QIAamp DNA Investigator kit (QIAgene, USA) according to the manufacturer’s instructions. Primers were designed on the basis of the sequence of the 28S regions of rDNA. The sequences of primers are as follows: 28S-F (50 -AAGCATATCAATAAGCGGAGG-30 ) as forward and 28S-R (50 -GGTCCGTGTTTCAAGACGG-30 ) as reverse. Polymerase chain reaction (PCR) was carried out in a thermal cycler (PeQLab, Germany) with the following temperature profile: 1 cycle of 5 min denaturation at 94 1C; 30 cycles of 35 s denaturation at 94 1C, primer annealing for 1 min at 53 1C, primer extension for 2 min at 72 1C; final 10 min extension at 72 1C. A negative control was also run along with the experiment. Each amplification products were loaded onto 1% agarose gel and run in TBE buffer (90 mM Tris, 90 mM boric acid and 2 mM EDTA, [pH 8.3]) at 80 V for 1 h. The products were detected by staining with ethidium bromide (0.5 mg/mL) and photographed. A single banding pattern with approximate size of 630 base pairs has been visualized after performing electrophoresis. This PCR product was then directly subjected for DNA sequencing. Analysis of the sequencing results demonstrated a high homology (99%) between the investigated amplicon and the 28S rDNA regions of A. candidus presented in the Gene data Bank (NCBI, NIH, USA). The obtained sequence of this gene fragment has been submitted to the gene date bank and registered under the accession number of JQ846017. It is noteworthy that treatment was immediately started according to the direct examination results and Terbinafine was prescribed in an oral dosage of 250 mg daily for six weeks. However, no remedy was obtained. After four months, a direct microscopic analysis was done for the second time and fungal filaments were again observed. Resistance to Terbinafine motivated us to perform antifungal drug susceptibility of the obtained isolate. E-test method was applied to determine the susceptibility of this clinical isolate to Itraconazole, Posaconazole, Fluconazole and Caspofungin (AB Biodisk, Sweden) as described in CLSI document M38. In the case of Terbinafine, microdilution method Fig. 3. Colony morphology of the causing agent of onychomycosis on Czapek Yeast Agar (CYA) after incubating at 251 C for 6 days. (a) The picture depicts a pale yellow colony. And the microscopic view of filaments after performing slide culture: (b) the fungus possesses white, typically globose, conidial heads producing globose or subglobose, smooth, thin-walled conidia with spherical vesicles and metulae cover the entire surface of the vesicle. (Scale bar: 2 mm.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). B. Ahmadi et al. / Medical Mycology Case Reports 1 (2012) 45–48 47 Fig. 4. After 72 h of incubation at 35 1C, it was found that the isolate was resistant to Caspofungin with MIC 432 mg/mL and moderately susceptible to Fluconazole with MIC 2 mg/mL. However it was susceptible to Posaconazole with MICs 0.032 mg/mL. Table 1 In vitro susceptibility of Aspergillus candidus against five antifungal drugs expressed in mg/ml. Itraconazole Fluconazole Psaconazole Caspofungin Terbinafine a b MIC (lg/mL) Drug breakpoints (lg/mL)a Methoda 0.064 2 0.032 4  32 Z  0.625 1 464 1 1 NDb E-test E-test E-test E-test Broth microdilution Methods used according to CLSI document M38-A2. Non-determined. was applied according to CLSI document M38. RPMI 1640 medium with L-glutamine and phenol red without bicarbonate and buffered to pH 7.0 at 25 1C was used as the culture medium. Terbinafine (Cipla, India) was dissolved in dimethylsulphoxide (DMSO) to 5 mg/mL, and then diluted to the final concentration of 0.009–5 mg/mL with the medium, according to the standard in the CLSI reference method. After growing on SC medium (Merck, Germany) at 35 1C for 7 days, fungal conidia were inoculated into a 96-well plate at a concentration of 106 cells/mL and incubated at 35 1C for 48 h. The MIC for the isolate against Terbinafine was determined as Z 0.625 mg/mL. To perform E-test for the other drugs, conidia from the newly identified A. candidus (approximately 106) were swabbed onto RPMI 1640 agar plates supplemented with 2% glucose, and the plate was allowed to be at room temperature for 10–15 min. E-test strips containing antifungal agents were applied and MICs were determined after 72 h incubation at 35 1C. The isolate was susceptible to Itraconazole, Posaconazole and Fluconazole with MICs of 0.064 mg/mL, 0.032 mg/mL and 2 mg/mL, respectively. But this isolate was resistant to Caspofungin with MIC of 432 mg/mL (Fig. 4). Table 1 shows the results for drug susceptibility test against the isolate of A. candidus using the above mentioned drugs. Oral Itraconazole was then prescribed immediately according to the drug susceptibility test with the dosage of 200 mg daily for 12 weeks. Ten weeks later, the patient was again referred to the medical mycology laboratory. Direct microscopic analysis was done and fungal filaments were not observed. 3. Discussion A. candidus is broadly distributed in nature and develops on vegetation in the later stages of decay [10]. This species is one of the most prevalent contaminant molds in cereal grains and flour [11,12]. A. candidus is considered as a potential respiratory hazard for grain workers even in immune competent individuals and a novel case of invasive aspergillosis caused by A. candidus has been reported [13]. Probable diseases in which A. candidus is claimed to be involved are invasive aspergillosis [13], otomycosis [14], brain granuloma [15]. In addition, allergic alveolitis or asthma, mycotoxicosis and related diseases, which are associated with substances released from both inhaled spores and mycelium fragments in the lungs, have also been reported to be triggered by A. candidus [16]. This fungus is considered as a rare pathogenic agent affecting the nail plate and a few case reports have been published worldwide [17–19]. Up to date, several Aspergillus species are responsible for onychomycosis including: A. versicolor, A. terreus, A. fumigatus, A. flavus and A. niger [5,20–23]; however, A. candidus is a rare cause of onychomycosis. Our probable diagnosis was suggested on the basis of several microscopic examination and positive cultures for A. candidus. Definitely, these methods of identification do not determine the causing agent of the disorder accurately. So, we benefited from PCR-based approaches to ensure about our identification. The result of molecular identification was consistent with our morphological identification and subsequently A. candidus was firmly introduced as the agent of this case of onychomycosis. Morphological identification is not sufficient for exact characterization of A. candidus due to several morphological similarities between this fungus and other Aspergillus species such as A. albus, A. okazakii, or A. dubius [10]. As the correct identification of the causing agent is important for the further assessment of drug susceptibility of the agent in order to choose an effective therapy, the molecular analysis of this isolate has been performed to validate the identification of A. candidus. Since the NDMs are not usually keratinolytic, they are usually regarded as the secondary invader of nail plate. Pre-existing disorder(s) like a certain disease or trauma may allocate the nail plate as a favorite location to invade [24]. But this is not always the case. As we mentioned in the case history, our patient neither had a known trauma or other predisposing factor, nor had an immune suppression disease. The only factor that our patient claimed about which may have increased the risk of getting affected by the fungus was working with water for a long period of time. Besides, the age of our patient (60 years) may also have probably played a role in getting affected with the infection. Unfortunately, a standard treatment for onychomycosis due to NDMs has not yet been established and perfect cure is rather difficult to obtain. According to CLSI M38-A2 document, the breakpoint for Terbinafine has not been established for molds testing yet. Nevertheless, application of oral Terbinafine, which is recommended for treatment of onychomycosis [5], was not effective in our patient. Therefore, it is strongly suggested that the 48 B. Ahmadi et al. / Medical Mycology Case Reports 1 (2012) 45–48 obtained isolate was resistant to Terbinafine. However, the isolated A. candidus was susceptible to Itraconazole and Posaconazole and the mentioned patient has been recovered successfully by applying Itraconazole. In conclusion, we described a case of onychomycosis caused by A. candidus in Iran and completely characterized the obtained fungus by both the conventional and molecular methods. Conflict of interest There are none. Acknowledgments The authors wish to thank Dr. Hamid Badali for helping us in performing drug susceptibility test. This research has been financially supported by the Tehran University of Medical sciences (TUMS) grant. References [1] Welsh O, Vera-Cabrera L, Welsh E. Onychomycosis. Clinics in Dermatology 2010;28(2):151–9. [2] Garcia-Martos P, Guarro J, Gené J, Mira J, Linares M, Ortoneda M. Onychomycosis caused by Aspergillus sclerotiorum. Journal de Mycologie Médicale 2001;11(4):222–4. [3] Chadeganipour M, Nilipour S, Ahmadi G. Study of onychomycosis in Isfahan, Iran. Mycoses 2010;53(2):153–7. [4] Gupta AK, Ryder JE, Summerbell RC. The diagnosis of nondermatophyte mold onychomycosis. International Journal of Dermatology 2003;42(4):272–3. [5] Tosti A, Piraccini BM, Lorenzi S. Onychomycosis caused by nondermatophytic molds: clinical features and response to treatment of 59 cases. Journal of the American Academy of Dermatology 2000;42(2):217–24. [6] Grover C, Khurana A. Onychomycosis: newer insights in pathogenesis and diagnosis. Indian Journal of Dermatology, Venereology and Leprology 2012;78(3): 263–70. [7] Singal A, Khanna D. Onychomycosis: diagnosis and management. Indian Journal of Dermatology, Venereology and Leprology 2011;77(6):659–72. [8] Moreno G, Arenas R. Other fungi causing onychomycosis. Clinics in Dermatology 2010;28:160–3. [9] de Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of clinical fungi. 2nd ed. Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures; 2000. [10] Raper K, Fennell D. The genus Aspergillus. Baltimore: Williams & Wilkins; 1965. [11] Taligoola H, Ismail M, Chebon S. Mycobiota associated with rice grains marketed in Uganda. Journal of Biological Sciences 2004;4(1):271–8. [12] Hocking AD, editor. Microbiological facts and fictions in grain storage. Canberra: CSIRO; 2003. [13] Ribeiro S, Santana A, Arriagada G, Martins J, Takagaki T. A novel cause of invasive pulmonary infection in an immunocompetent patient: Aspergillus candidus. Journal of Infection 2005;51(4):e195–7. [14] Yassin A, Maher A, Moawad M. Otomycosis: a survey in the eastern province of Saudi Arabia. Journal of Laryngology and Otology 1978;92(10):869–76. [15] Linares G, McGarry PA, Baker RD. Solid solitary aspergillotic granuloma of the brain. Report of a case due to Aspergillus candidus and review of the literature. Neurology 1971;21(2):177–84. [16] Krysińska-Traczyk E, Dutkiewicz J. Aspergillus candidus: a respiratory hazard associated with grain dust. Annals of Agricultural and Environmental Medicine 2000;7(2):101–9. [17] Cornere B, Eastman M. Onychomycosis due to Aspergillus candidus: case report. New Zealand Medical Journal 1975;82(543):13. [18] Fragner P, Kubickova V. Onychomycosis due to Aspergillus candidus. Ceskoslovenska Dermatologie 1974;49(5):322–4. [19] Kaben U. Aspergillus candidus link as the cause of onychomycosis. Zeitschrift fur Haut- und Geschlechtskrankheiten 1962;32:50–3. [20] Gupta AK, Ryder JE, Baran R, Summerbell RC. Non-dermatophyte onychomycosis. Dermatologic Clinics 2003;21(2):257. [21] Torres-Rodrı́guez J, Madrenys-Brunet N, Siddat M, López-Jodra O, Jimenez T. Aspergillus versicolor as cause of onychomycosis: report of 12 cases and susceptibility testing to antifungal drugs. Journal of the European Academy of Dermatology and Venereology 1998;11(1):25–31. [22] Torres-Rodriguez J, Balaguer-Meler J, Martinez A, Antonell-Reixach J. Onychomycosis due to a fungus of the Aspergillus versicolor group. Mycoses 1988;31(11):579–83. [23] Tosti A, Piraccini BM. Proximal subungual onychomycosis due to Aspergillus niger: report of two cases. British Journal of Dermatology 1998;139(1): 156–7. [24] Eenglish MP. Nails and fungi. British Journal of Dermatology 1976;94(6): 697–701.